Pseudomonas aeruginosa is an opportunistic pathogen that can infect many different tissues. Similar to what has been observed in other Gram-negative bacteria, one of the virulence factors important for acute P. aeruginosa infections is lipopolysaccharide (LPS). In the case of P. aeruginosa, the O antigen portion is thought to be essential for systemic infections. Chronic lung infections in cystic fibrosis patients, on the other hand, are caused by P. aeruginosa strains that have a defective LPS with few or no O antigens; these infections remain localized to the lung. In other P. aeruginosa respiratory infections dissemination to the blood is also rare, suggesting that O antigen expression, which confers resistance to the action of normal human serum, may not be required for infections localized to the lung. Prior evaluations of P. aeruginosa LPS mutants in various infection models used either genetically or structurally undefined strains, or those with pleiotropic effects. Therefore the question as to the role of O antigen in P. aeruginosa pathogenesis clearly needs to be reevaluated. The objective of this proposal is to determine which portions of the LPS are critical for infections at particular sites. The genetic locus encoding the enzymes for the synthesis of P. aeruginosa serogroup O11 O antigen has been cloned from strain PA103. Previous work from a number of laboratories has shown PA103 to be highly virulent in many animal models of infection. The steps in the pathway of synthesis of this O antigen will be characterized through mutational, structural, and biochemical analysis of the genes of the O antigen locus (Specific Aim 1). The transcriptional organization of the O antigen gene locus will also be determined (Specific Aim 2). In order to ascertain which portions of the P. aeruginosa LPS are required for virulence, the genetically and structurally defined LPS mutants constructed here will be tested in two different mouse models of infection. In the intranasal infection model, infections can remain localized to the lung or can translocate from the lungs to the blood and other organs. In the corneal infection model, the P. aeruginosa infection remains localized to the eye after trauma (Specific Aim 3). The long-term goal of this research is to devise rational strategies to target LPS synthetic enzymes for the development of vaccines and antibacterial agents to inhibit infections caused by this important human pathogen.